Quantitative determination method for chloride ions

ABSTRACT

Chloride ion is quantitatively determined in a sample by adding to the sample an enzyme having glucokinase activity to thereby eliminate glucose, admixing an α-amylase which has been deactivated by a chelating agent, and reacting the α-amylase with maltotetraose, maltohexaose or maltoheptaose.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a quantitative determination method forchloride ions in an organism which is applicable to clinical tests.

2. Description of the Prior Art

There is known a quantitative determination method for chloride ionscomprising reacting an α-amylase with a chloride ion-containing sampleand a substrate, such as p-nitrophenylmaltoside or starch, by utilizingthe phenomenon that an α-amylase deactivated by a chelating agent isactivated by chloride ions Eur. J. Biochem. 41: 171 (1974)!. There isknown another quantitative determination method for chloride ionswherein 2-chloro-4-nitrophenyl-β-D-maltoheptaoside is used as asubstrate in order to simplify the process for measuring the α-amylaseactivity in the above quantitative determination method Clin. Chem., 34:552 (1988)!.

As a method for measuring the activity of an α-amylase, there is known amethod comprising producing maltose by using an oligosaccharide as asubstrate, converting the resultant maltose into glucose andquantitatively determining the glucose obtained to thereby measure thecorresponding α-amylase activity J. Clin. Chem. Clin. Biochem., 17: 705(1979)!.

Since glucose coexisting in a sample influences a quantitativedetermination method for a substance in a sample, a method is disclosedin Japanese Unexamined Patent Publication No. 5-76397 wherein glucose ina sample is eliminated by using hexokinase or the like.

Out of the quantitative determination methods for chloride ions, themethod using a synthetic substrate such as2-chloro-4-nitrophenyl-β-D-maltoheptaoside for measuring the activity ofan α-amylase must use the two-point calibration method because linearityin calibration curves is hard to obtain. The known method by usingstarch as a substrate for an α-amylase reaction and measuring thereaction product, i.e. a reducing sugar such as glucose and maltose,cannot accurately determine the amount of chloride ions when a bloodsample is used, because glucose and maltose are present in blood.Therefore, if a method of newly using a maltooligosaccharide as a usefulsubstrate for an α-amylase reaction is employed, it will be impossibleto achieve an accurate determination because of the interference ofglucose and the like present in blood.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a quantitative determinationmethod for chloride ions which can accurately determine the amount ofchloride ions even when blood is used as a sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a calibration curve using maltopentaose as a substrate.

FIG. 2 is a calibration curve using maltotetraose as a substrate.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The method of the invention for quantitative determination of chlorideions relates to a method of determining, in an aqueous medium, chlorideions in a sample by using an α-amylase which has been deactivated by achelating agent. The method of the present invention is characterized byadding to a sample in advance adenosine triphosphate and an enzymehaving glucokinase activity to thereby eliminate glucose in the sample,deactivating the enzyme having glucokinase activity and determining theamount of glucose produced by a reaction of α-amylase activated by thechloride ions using an oligosaccharide as a substrate for the reaction.

The method of the invention has been achieved based on the followingfinding that, in the above method of determining in an aqueous mediumchloride ions in a sample by using an α-amylase which has beendeactivated by a chelating agent, the sample is pretreated, theoligosaccharide is used as substrate and the α-amylase activityactivated by the chloride ion is determined by measuring the amount ofglucose as a final product, the amount of chloride ions can beaccurately determined. In particular, the method for the pretreatment ofa sample has been established based on the following finding that a moreaccurate determination of chloride ions can be achieved by adding, to asample, not only an enzyme having glucokinase activity for eliminationof glucose in a sample but also an enzyme-inhibitor in order to preventthe enzyme having glucokinase activity from interfering the glucosedetermination system.

The method for the pretreatment of a sample as described above compriseseliminating glucose in a sample by adding to the sample in advanceadenosine triphosphate and an enzyme having glucokinase activity andthen deactivating the enzyme having glucokinase activity with aninhibitor of the enzyme such as a chelating agent or the like. As aresult of the pretreatment, glucose which interferes with thequantitative determination of chloride ions is removed.

Further, in the present invention, there is provided a quantitativedetermination method for chloride ions which is not influenced even whenmaltose coexists in a sample, the method being achieved by appropriatelyselecting the kind of α-amylase and the kind of oligosaccharide as asubstrate thereof.

In the present invention, the method of determining, in an aqueousmedium, chloride ions in a sample by using an α-amylase which has beendeactivated by a chelating agent means a method wherein, in an aqueousmedium, an α-amylase which has been deactivated by mixing with achelating agent and is re-activated by the chloride ions in a sampledecomposes the substrate and the amount of a decomposed-product ismeasured to determine the amount of the corresponding chloride ions.

In the present invention, "an aqueous medium" means a liquid containingwater, such as buffer and physiological saline. As examples of thebuffer, tris(hydroxymethyl)aminomethane-nitrate buffer,tris(hydroxymethyl)aminomethane-sulfate buffer, phosphate buffer,acetate buffer, succinate buffer, phthalate buffer, borate buffer,glycine buffer, barbital buffer, Good's buffer and the like may beenumerated. The pH of the buffer is 6-9.5 and the concentration is50-500 mM.

As a sample containing chloride ions, any sample may be used as long asit is miscible with an aqueous medium. For example, biosamples such aswhole blood and cells, though which are difficult to quantitativelydetermine by the ion electrode method may be enumerated.

As an α-amylase, any enzyme belonging to the enzyme number EC. 3.2.1.1may be used. Animal-derived α-amylases taken from porcine pancreas,human saliva and the like or enzymes produced by modifying thoseamylases by genetic engineering techniques may be enumerated. In thepresent invention, it is preferably to use a porcine pancreas-derivedα-amylase because, when maltoheptaose, maltohexaose or maltotetraose isused as an oligosaccharide substrate, the enzyme decomposes thesubstrate into only maltotriose and glucose, and not into maltose; thus,an elimination operation for maltose is not required.

As a chelating agent, ethylenediaminetetraacetic acid (EDTA),2-hydroxyethylethylenediaminetriacetic acid (HEDTA), ethyleneglycol-bis(2-aminoethyl ether) tetraacetic acid (EGTA),diethylenetriaminepentaacetic acid (DTPA), 1,2-diaminocyclohexanetetraacetic acid (DCTA) and the like may beenumerated.

As an oligosaccharide used as a substrate for an α-amylase,maltooctaose, maltoheptaose, maltohexaose, maltopentaose, maltotetraoseand the like may be enumerated. When an oligosaccharide is decomposed byan α-amylase to produce maltose as well as glucose, an enzyme of whichthe substrate is maltose may be added, if necessary, to thereby convertmaltose into glucose. Among oligosaccharides, maltoheptaose,maltohexaose and maltotetraose are selectively decomposed into glucoseby a porcine pancreas-derived α-amylase and are not decomposed intoglucose by a human amylase contaminated in a sample. Therefore, use ofthese oligosaccharides is preferable in the present invention.

As to a quantitative determination method for glucose, any method fordetermining the amount glucose may be used. For example, a methodwherein glucose is converted into glucosone or gluconic lactone bypyranose oxidase or glucose oxidase, respectively, and the hydrogenperoxide generated is quantitatively determined with a peroxidase and acoloring reagent Ann. Clin. Biochem., 6: 24 (1969)! or a method whereinglucose is converted into gluconic lactone by glucose dehydrogenase andthe amount converted from NAD(P) to NAD(P)H is determined Z. Kin. Chem.Klin. Biochem., 13: 101 (1975)! may be used.

As a coloring reagent, for example, a combination of 4-aminoantipyrinewith N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine (EMSA) or thelike may be used.

In the quantitative determination method for glucose mentioned above,when endogenous glucose or maltose coexists in a sample, such glucose ormaltose gives an influence upon the determined value of chloride ions.Therefore, as a pretreatment process for the above determination method,adenosine triphosphate, an enzyme having glucokinase activity and, ifnecessary, the above-mentioned enzyme of which the substrate is maltoseare added to a sample in advance to thereby eliminate glucose or maltosein a sample and then a chelating agent is added to the sample todeactivate the enzyme having glucokinase activity so that this enzymecoexisting in the sample does not inhibit the process of quantitativedetermination of chloride ions.

As an enzyme having glucokinase activity, any enzyme may be used as longas it has an activity to convert glucose into glucose-6-phosphoric acid.For example, hexokinase (EC. 2.7.1.1) derived from animals (such aserythrocytes and a liver) or microorganisms, hexokinase type IV (EC.2.7.1.2) derived from animals (such as a liver) or microorganisms (suchas bacteria and yeast) and the like may be enumerated.

As a chelating agent which is used for deactivating an enzyme havingglucokinase activity, chelating agents similar to those used fordeactivating an α-amylase may be used. For example, EDTA, HEDTA, EGTA,DTPA, DCTA and the like may be enumerated.

As an enzyme of which the substrate is maltose, any enzyme which canconvert maltose into glucose, such as maltose phosphorylase (EC.2.4.1.8) or α-glucosidase (EC. 3.2.1.20), may be used. When maltosephosphorylase is used, phosphate ions are added to a reaction solutiontogether with this enzyme if phosphate buffer is not used. As a sourceof these phosphate ions, inorganic phosphoric acid, monopotassiumphosphate, dipotassium phosphate, monosodium phosphate, disodiumphosphate and the like may be enumerated. It should be noted that when aporcine pancreas-derived α-amylase is used together with maltoheptaose,maltohexaose or maltotetraose as a substrate, it is not necessary to addan enzyme of which the substrate is maltose because maltose will not begenerated.

Preferred embodiments of the quantitative determination method of theinvention for chloride ions will be described below.

To an aqueous medium (pH 6-9.5), an enzyme having glucokinase activity(1-4 U/ml), adenosine triphosphate (ATP)(5-20 mM), magnesium ions (2-25mM) and, if necessary, an enzyme of which the substrate is maltose (whenmaltose phosphorylase is used, 4-20 U/ml of this enzyme and 300 mM ormore of phosphoric acid are added against 1 g/dl of maltose; whenα-glucosidase is used, 100-300 U/ml of this enzyme is added against 1g/dl of maltose)are added to prepare a pretreatment solution. A samplecontaining chloride ions is added to the pretreatment solution and thenreacted at 25-40° C., preferably 37° C., for 1-30 minutes, preferably3-5 minutes.

An oligosaccharide (2-10 mM in the reaction solution) may be added tothe pretreatment solution in advance or may be added after thecompletion of the pretreatment reaction. Further, a chelating agent(10-50 mM in the reaction solution) and an α-amylase which has beendeactivated by a chelating agent (20-100 U/ml in the reaction solution)are added to the solution and reacted at 8-50° C. The oligosaccharide(the substrate) is decomposed into maltose, glucose and maltotriosethrough an α-amylase reaction. When maltose is generated, the maltose isdecomposed into glucose by the above-mentioned enzyme of which thesubstrate is maltose. By determining the amount of the resultant glucoseby the method described above, the amount of the corresponding chlorideions can be determined. A chelating agent which inhibits glucokinaseactivity also deactivates α-amylase. Therefore, if a solution isprepared in advance by adding an α-amylase (20-100 U/ml in the reactionsolution) and reagents for the quantitative determination of glucosesuch as glucose oxidase and EMSA to an aqueous medium containing achelating agent (10-50 mM in the reaction solution) and the preparedsolution is added to the pretreatment solution for a sample, the amountof chloride ions can be simply determined.

To the reaction solution, there may be added albumin, flavin adeninedinucleotide (FAD) which is a coenzyme of pyranose oxidase, glycerol, achloride ion-free surfactant such as polyethylene glycolmono-p-isooctylphenyl ether and condensation products of polyoxyethyleneand polyoxypropylene, a solubilizer such as sodium nitrate and sodiumsulfate, and stabilizer such as potassium nitrate and potassium sulfate.Further, by adding glucose (0.08-0.2 mg/ml) to the pretreatmentsolution, a blank absorption can be eliminated.

With respect to the enzymes used in the present invention, for example,maltose phosphorylase (EC. 2.4.1.8), α-glucosidase (EC. 3.2.1.20) andhexokinase (EC. 2.7.1.1) or hexokinase type IV glucokinase (EC.2.7.1.2)! as an enzyme having glucokinase activity, commercial productsare easily available for each of them.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing a calibration curve obtained by usingmaltopentaose as a substrate. FIG. 2 is a graph showing a calibrationcurve obtained by using maltotetraose as a substrate.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described in more detail with reference tothe following Examples, which should not be construed as limiting thescope of the invention.

EXAMPLE 1 A Method using a Porcine Pancreas α-amylase and Maltopentaose

(1) Preparation of Standard Solutions for Obtaining a Chloride IonCalibration Curve

Sodium chloride (Wako Pure Chemicals) was diluted with distilled waterto prepare standard solutions for obtaining a chloride ion calibrationcurve so that the final concentrations in the reaction solution become60, 100 and 140 mM.

(2) Quantitative Determination of Chloride Ions

To a test tube, 0.10 ml of the standard solution for obtaining achloride ion calibration curve, 0.10 ml of an aqueous glucose solution(1 g/dl) and 0.10 ml of an aqueous maltose solution (1 g/dl) were added.Then, 2.0 ml of 350 mM phosphate buffer (pH 6.8, 25° C.) containing 7U/ml maltose phosphorylase (Kyowa Medex), 2.4 U/ml hexokinase (OrientalYeast), 10 mM maltopentaose (Kyowa Medex), 0.5 mM 4-aminoantipyrine, 2.5mM magnesium sulfate and 10 mM ATP (Oriental Yeast) was added thereto.The test tube was put in a constant-temperature water bath at 37° C. andincubated for 5 minutes, to thereby eliminate maltose and glucose.

Thereafter, to a solution (25° C.) containing 20 g/l monopotassiumphosphate and 18 g/l EDTA, of which the pH has been adjusted to 6.8 withsodium hydroxide, a coloring solution containing 50 U/ml porcinepancreas α-amylase (Boehringer Mannheim Yamanouchi), 30 U/ml glucoseoxidase (Wako Pure Chemicals), 2.2 mM EMSA and 1 mM potassium nitratewas added and incubated at 37° C. Then, 1 ml of this solution was addedto the solution prepared above. After stirring, changes in absorbance at555 nm were measured with a spectrophotometer (Model UV3400, Hitachi).The calibration curve obtained is shown in FIG. 1.

EXAMPLE 2 A Method using a Porcine Pancreas α-amylase and Maltotetraose

(1) Preparation of Standard Solutions for Obtaining a Chloride IonCalibration Curve

Sodium chloride (Wako Pure Chemicals) was diluted with distilled waterto prepare standard solutions for obtaining a chloride ion calibrationcurve so that the final concentrations in the reaction solution become50, 100 and 150 mM.

(2) Quantitative Determination of Chloride Ions

To a test tube, 0.05 ml of the standard solution for obtaining achloride ion calibration curve, 0.05 ml of an aqueous glucose solution(1 g/dl) and 0.05 ml of an aqueous maltose solution (2 g/dl) were added.Then, 2.0 ml of 100 mM phosphate buffer (pH 6.6, 25° C.) containing 2.4U/ml hexokinase (Oriental Yeast), 7.5 mM maltotetraose (Wako PureChemicals), 0.5 mM 4-aminoantipyrine, 5 mM magnesium sulfate and 10 mMATP (Oriental Yeast) was added thereto and incubated at 37° C. for 5minutes, to thereby eliminate glucose.

Thereafter, to a solution (25° C.) containing 20 g/l monopotassiumphosphate and 36 g/l EDTA, of which the pH has been adjusted to 6.6 withsodium hydroxide, a coloring solution containing 70 U/ml porcinepancreas α-amylase (Boehringer Mannheim Yamanouchi), 30 U/ml glucoseoxidase (Wako Pure Chemicals), 2.2 mM EMSA and 1 mM potassium nitratewas added and incubated at 37° C. Then, 1 ml of this solution was addedto the solution prepared above. After stirring, changes in absorbance at555 nm were measured with a spectrophotometer (Model UV3400, Hitachi).The calibration curve obtained is shown in FIG. 2. In addition, a serumwhose chloride ion concentration was 105 mM as determined by coulometrictitration was measured for its chloride ion concentration according tothe above method. As a result, the chloride ion concentration was found104 mM.

According to FIG. 2, the calibration curve passes through the origin andthus it is possible to carry out a one-point calibration. Further, ithas been demonstrated that the method of the invention is an excellentdetermination method because; the absorption of blank is little and themethod is not influenced by maltose nor endogenous, saliva-derived orpancreas-derived amylase in a sample.

According to the present invention, a quantitative determination methodfor chloride ions is provided which is not influenced by glucose,maltose and the like coexisting in a sample and is excellent indetermination accuracy. The quantitative determination method of theinvention for chloride ions is useful as a clinical test.

We claim:
 1. A method for quantitatively determining chloride ion in asample, comprising the steps of:(1) admixing (i) an enzyme having aglucokinase activity, (ii) adenosine triphosphate and (iii) an aqueousmedium comprising said sample; (2) reacting said sample with said enzymeto eliminate any glucose coexisting in said sample, to obtain mixture(A); (3) admixing mixture (A) with (i) an α-amylase which has beendeactivated by a chelating agent and (ii) a chelating agent todeactivate the enzyme having a glucokinase activity, to obtain mixture(B); (4) reacting said α-amylase of mixture (B) with a naturallyoccurring oligosaccharide selected from the group consisting ofmaltotetraose, maltohexaose and maltoheptaose; (5) determining an amountof glucose produced in the reaction of step (4); and (6) correlatingsaid amount of glucose with a quantity of chloride ion in said sample.2. The method of claim 1, wherein said α-amylase is derived from porcinepancreas.
 3. The method of claim 1, wherein said enzyme having aglucokinase activity is hexokinase.
 4. The method of claim 4, whereinsaid enzyme having a glucokinase activity is hexokinase type IV.
 5. Themethod of claim 1, wherein chloride ion is determined without usingtwo-point calibration.
 6. The method of claim 1, wherein a concentrationof said chelating agent in step (3) is 10-50 mM.
 7. The method of claim1, wherein said chelating agent is selected from the group consisting ofethylenediaminetetraacetic acid (EDTA),2-hydroxyethylethylenediaminetriacetic acid (HEDTA), ethyleneglycol-bis(2-aminoethyl ether)tetraacetic acid (EGTA),diethylenetriaminepentaacetic acid (DTPA) and1,2-diaminocyclohexanetetraacetic acid (DCTA).
 8. The method of claim 1,wherein said oligosaccharide is added to mixture (B) in a concentrationof 2-10 mM.
 9. The method of any one of claims 1-8, wherein glucose iscorrelated with chloride ion using a calibration curve thatsubstantially passes through its origin.